Methods for prenatal diagnosis of chromosomal abnormalities

ABSTRACT

Chromosomal abnormalities are responsible for a significant number of birth defects, including mental retardation. The present invention is related to methods for non-invasive and rapid, prenatal diagnosis of chromosomal abnormalities based on analysis of a maternal blood sample. The invention exploits the differences in DNA between the mother and fetus, for instance differences in their methylation states, as a means to enrich for fetal DNA in maternal plasma sample. The methods described herein can be used to detect chromosomal DNA deletions and duplications. In a preferred embodiment, the methods are used to diagnose chromosomal aneuploidy and related disorders, such as Down&#39;s and Turner&#39;s Syndrome.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims benefit under 35 USC 119(e) of the U.S.provisional application No. 60/509,775 filed on Oct. 8, 2003, thecontent of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is related to methods for non-invasive, prenataldiagnosis of chromosomal abnormalities. The methods of the invention canbe used to detect chromosomal DNA deletions and duplications. In apreferred embodiment, the methods are used to diagnose chromosomalaneuploidy and related disorders, such as Down's and Turner's Syndrome.The present invention further provides for methods of identifyingmethyl-polymorphic probes that can be used for the detection of fetalchromosome abnormalities.

BACKGROUND OF THE INVENTION

Chromosomal abnormalities are responsible for a significant number ofbirth defects, including mental retardation. Abnormalities can appear inthe form of chromosomal DNA duplications and deletions, as well in theform as chromosomal aneuploidy, which is the abnormal presence orabsence of an entire chromosome. Conditions where an organism has lessthan, or more than the normal diploid number of chromosomes give rise toa multitude of abnormal characteristics and are responsible for manysyndromes. Down's syndrome, or trisomy 21, is the most common example ofa chromosomal aneuploidy and involves an extra chromosome 21. Othercommon chromosomal aneuploidies are trisomy 13, trisomy 18, Turner'ssyndrome and Klinefelter's syndrome.

The options for the prenatal detection of chromosomal abnormalities aremainly limited to invasive methods with a small but finite risk forfetal loss. The most common method for detection of abnormalities isamniocentesis. However, because amniocentisis is an invasive method itis generally performed only on older mothers where the risk of a fetuspresenting with chromosomal abnormalities is increased. It wouldtherefore be beneficial to establish non-invasive methods for thediagnosis of fetal chromosomal abnormalities that can be used on largerpopulation of prospective mothers. One such non-invasive method has beendescribed in U.S. Pat. No. 4,874,693, which discloses a method fordetecting placental dysfunction indicative of chromosomal abnormalitiesby monitoring the maternal levels of human chorionic gonadotropinhormone (HCG). However, while this method is non-invasive and can beused to screen prospective mothers of all ages, it does not serve as adiagnostic of the particular chromosomal abnormality present, nor is aguarantee of its presence.

In addition to being invasive at the sample taking step, the existingprenatal diagnosis methods are also time consuming to perform. Forexample, Geisma-staining is the technique most widely used and requiresthat the cells be in metaphase or dividing, when the test is preformed.Each chromosome pair stains in a characteristic pattern of light anddark bands. Using this method all of the chromosomes can be individuallydistinguished and readily reveal the nature of any structural ornumerical abnormalities. Geisma-staining does not always detect subtlechromosomal rearrangements. If chromosomal rearrangements are suspectedand not detected using this method, further detailed analysis can bedone using fluorescent in situ hybridization (FISH) or spectralkaryotyping (SKY). Tests results using Geisma-staining can take one totwo weeks.

SKY is a technique that paints each of the metaphase chromosomes with adifferent probe (dye color). Because each chromosome-specific probeemits its own signature wavelength of fluorescence, structuralrearrangement are easily seen, and the chromosomes involved can bereadily identified. SKY requires that cells be in metaphase thereforeresults can take one to two weeks.

FISH is a technique that uses a fluorescent probe (dye) that attaches,or hybridizes, to specific individual chromosomes or certain regions ofchromosomes. The affected chromosomes or regions fluoresce, or signal,their presence, or lack of, and can be visually analyzed through afluorescent microscope. FISH is used to identify particular chromosomalrearrangements or rapidly diagnose the existence of an abnormal numberof chromosomes. FISH is currently the most rapid diagnosis method ofabnormal chromosome numbers. The speed is possible because cells do notneed to be in metaphase in order to do the analysis. Results of the testare typically known in two to three days

Thus, there is a need in the art for non-invasive prenatal diagnosticmethods that can rapidly and accurately help determine the presence andthe type of chromosomal aberrations.

SUMMARY OF THE INVENTION

The present invention describes a method for non-invasive prenataldiagnosis of chromosomal abnormalities, such as chromosomal aneuploidy,and allows rapid production of accurate results. The methods of theinvention use plasma samples obtained from a pregnant female. It hasbeen shown that maternal samples contain a small percentage of fetal DNAbut the percentage of the fetal cells present in the maternal plasma issmall.

The autosomal chromosomes have one allele inherited from the mother (A,as shown in the table below) and one allele from the father (B as shownin the table below). In a situation, wherein fetal DNA represents about2% of the total DNA present in the maternal plasma sample, the presenceof fetal alleles can be presented as follows: Maternal DNA Fetal DNA(98%) (2%) B % Trisomy of maternally AA AAB 2/202 inherited allele (A)Normal AA AB 2/200

Thus, because the difference of B % between normal and trisomy is only(2/200-2/202) or 0.01%, the difference is too small to detect using eventhe best available quantification methods.

The present invention solves this problem by enriching, relatively, theamount of fetal DNA in the maternal plasma sample before detecting thealleles present in the sample. To enrich for fetal DNA present in plasmaof the mother to allow accurate detection of fetal alleles present inthe sample, the invention exploits differences in the DNA between themother and fetus, for instance, differences in the DNA methylationstates. Thus, the maternal DNA can be substantially reduced, masked, ordestroyed completely, and the sample is left with DNA majority of whichis of fetal origin. The selective destruction of maternal DNA can beperformed using one or more enzymes, such as methylation sensitiveenzymes, which selectively digest maternal nucleic acids around theregion, which is later used for detection of the allele frequency. Theallele frequency of fetal DNA is then determined using polymorphicmarkers adjacent to the selected chromosomal regions. A difference inallele frequency as compared to a control sample is indicative of achromosomal abnormality.

In one embodiment, a method for detecting a chromosomal abnormality isprovided that comprises: a) obtaining a plasma sample from a pregnantfemale, b) optionally isolating DNA from the said plasma sample, c)digesting the DNA with an enzyme, such as a methyl-sensitive enzyme,that selectively digests the maternal or fetal DNA, d) using theselective digestion to obtain a DNA sample enriched for fetal ormaternal DNA, e) determining the maternal or paternal allele frequencyusing polymorphic markers adjacent to the selected fetal DNA regions,and f) comparing the paternal or maternal allele frequency of step e) toa control DNA sample, wherein a difference in allele frequency isindicative of a chromosomal abnormality. Preferably, one would alsocompare the putative abnormal DNA against a panel of normal DNA and/orabnormal DNA to take polymorphic differences into account.

Thus, if the maternal DNA is completely destroyed by digestion, thefetal allele frequency can be detected as shown in the table below:Maternal DNA in plasma sample Fetal DNA (100% in (digested, 0%) theplasma sample) B % Trisomy of AA AAB ⅓ maternally (or 33.3%) inheritedallele Normal AA AB ½ (or 50%)

The relative enrichment of the fetal DNA in the maternal plasma samplenow allows accurate detection of allele frequencies using practicallyany method of nucleic acid detection. The ratio between the maternal andpaternal allele in the maternal plasma sample thus reflects the allelicratio in the fetus only. Therefore, if more than two maternal allelesare present in the sample, the ratio will be significantly altered fromthe normal ½.

Any differences between the fetal and maternal DNA can be exploited, forexample exploitation of Y-chromosome specific DNA and telomere length.Differences in DNA between mother and fetus can be determined by knownmeans. In the case where the difference is differential methylation,methyl-sensitive enzymes digest unmethylated maternal DNA that ismethylated in the fetus, or vise versa. For instance, when the fetal DNAregion is methylated, methyl-sensitive enzymes are used to digestunmethylated maternal DNA. The digestion leaves only methylated fetalDNA fragments, thereby enriching for fetal DNA. Polymorphic markers thatare close to or within the differentially methylated DNA regions arethen used as labels to detect the frequency of maternal or paternal DNAin the maternal plasma sample. The allele frequency of the maternal andpaternal DNA is compared to the allele frequency that is normallyobserved in genomic DNA obtained from a healthy individual that does nothave a chromosome abnormality. In this manner, any chromosomalabnormalities can be detected. One or more alleles can be detectedsimultaneously, thus allowing screening of several chromosomalabnormalities simultaneously from the same sample. Alternatively,enzymes that digest only methylated DNA can be used to enrich for DNAthat is unmethylated in the fetus but methylated in the mother. Themethods of the present invention are suitable to detect chromosomal DNAduplications, or deletions, and to detect chromosomal aneuploidy.

While it is preferred that the first step destroys the maternal allelessubstantially completely, this is not necessary. The present inventionalso provides a method, wherein, if the maternal DNA is not completelydestroyed, a control allele is used from one or more chromosomes thatare not expected to be present in duplicate. The situation can bepresented as follows: Maternal Fetal B (or D) % B (or D) % DNA DNA 100%digestion 98% digestion Trisomy AA AAB 33.3% 20% Non-aneuploidy CC CD  50% 25%

In the table, the alleles B and D are paternally inherited allelespresent in the fetal DNA.

Alternatively, fetal DNA can be amplified further after the firstdigestion. Thus, the invention provides a method, wherein after theinitial digestion of maternal DNA, the sample is amplified using anamplification method which selectively amplifies the fetal DNA.Alternatively, one preserves the differences, for example themethylation differences, between the maternal and fetal DNA. Theamplified sample is consequently digested again thus allowing a largerpercentage of fetal DNA to be achieved. The digestion/amplificationscheme can be performed, of course, more than once, if desired.Amplification step can also be used together with a detection of acontrol allele.

Therefore, in one embodiment, the methods of the present inventionfurther comprise an amplification scheme to enrich for fetal DNA. In oneaspect, the amplification method comprises a) obtaining a plasma samplefrom a pregnant female and optionally isolating DNA from said sample, b)digesting isolated DNA with a methyl-sensitive enzyme that digests onlyunmethylated DNA, c) isolating undigested DNA from step b, d) amplifyingthe undigested DNA from step c while simultaneously using a DNAmethylase to methylate nascent hemi-methylated DNA, e) digestingamplified DNA of step c with an enzyme that digests only unmethylatedDNA, f) determining the maternal or paternal allele frequency using, forexample, polymorphic markers adjacent to methylated fetal DNA regionsand, g) comparing the paternal or maternal allele frequency of step f)to a control DNA sample, wherein a difference allele frequency isindicative of a chromosomal abnormality.

In another embodiment, the invention provides a method for the diagnosisof trisomy 21 (Down's syndrome). The method comprises: a) obtaining aplasma sample from a pregnant female, b) optionally isolating DNA fromsaid plasma sample, c) digesting the DNA with an enzyme, such as amethyl-sensitive enzyme, that digests only maternal or fetal DNA, d)determining the paternal allele frequency using polymorphic markersadjacent to the selected fetal DNA regions of chromosome 21, and e)comparing the paternal allele frequency of step d to a control DNAsample, wherein a paternal allele frequency less than the control isindicative of Downs's syndrome.

In another embodiment, the invention provides a kit for detectingchromosomal aneuploidy in the maternal plasma sample, wherein the kitcomprises one or more enzymes to specifically digest the maternal DNA inthe plasma sample of a pregnant female, and primers to detect paternaland maternal allele frequency of polymorphic markers in the enrichedfetal DNA regions in order to detect chromosomal deletions, insertionsor aneuploidy. The kit may also comprise containers, enzymes, such aspolymerases, and buffers to facilitate the isolation of nucleic acidsfrom the maternal plasma sample, and amplification of markers to detectthe allele frequency. The kit may also contain standard or control DNAs,such as DNA isolated from plasma of a mother pregnant with a healthyfetus, and/or DNA samples isolated from plasma samples from femalescarrying a fetuses with chromosomal abnormalities such as chromosome 21,13, and/or 18 trisomy.

A kit for prenatal diagnosis of chromosomal abnormalities preferablycomprises at least one methylation-sensitive enzyme, at least one pairof nucleic acid amplification primers capable of annealing and thusamplifying regions flanking sites that contain at least one polymorphiclocus within differentially methylated regions in fetal and maternal DNApresent in maternal plasma, at least one primer or probe to allowdetection of alleles in the at least one polymorphic locus, and aninstruction manual instructing the user to perform the steps of taking aplasma sample from a pregnant female, selectively digesting the nucleicacids present in said plasma sample with the methylation-sensitiveenzyme to enrich the fetal nucleic acids in the sample, performingnucleic acid amplification using the amplification primers and detectingthe alleles present in the sample enriched for the fetal nucleic acids,and interpreting the results so that if the ratio of two differentalleles in the locus deviates from a control wherein the alleles arepresent in equal amounts, the fetus is affected with a chromosomalabnormality.

The kit may further comprise a control nucleic acid panel, wherein thecontrols comprise nucleic acids isolated from females pregnant withfetuses carrying known chromosomal abnormalities and females pregnantwith fetuses without chromosomal abnormalities.

The kit may also further comprise an internal control of at least onepair of amplification primers and a detection primer or probe, whereinthe primers and/or probe are selected from a nucleic acid region that isdifferentially methylated in fetal and maternal DNA present in maternalplasma, but that occur in chromosomes, wherein duplication or deletionis rare, so as to provide an internal control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for the detection of fetalchromosome abnormalities.

As used herein, the term “chromosomal abnormality” refers to achromosome with DNA deletions or duplications and to chromosomalaneuploidy. The term also encompasses translocation of extra chromosomalsequences to other chromosomes.

As used herein, the term “chromosomal aneuploidy” refers to the abnormalpresence (hyperploidy) or absence (hypoploidy) of a chromosome.

As used herein, the term “polymorphic marker” refers to segments ofgenomic DNA that exhibit heritable variation in a DNA sequence betweenindividuals. Such markers include, but are not limited to, singlenucleotide polymorphisms (SNPs), restriction fragment lengthpolymorphisms (RFLPs), short tandem repeats, such as di-, tri- ortetra-nucleotide repeats (STRs), and the like. Polymorphic markersaccording to the present invention can be used to specificallydifferentiate between a maternal and paternal allele in the enrichedfetal nucleic acid sample.

As used herein, the term “methyl-polymorphic marker” refers to apolymorphic marker that is adjacent to differentially methylated DNAregions of fetal and maternal DNA. The term adjacent refers to a markerthat is within 1-3000 base pairs, preferably 1000 base pairs, morepreferably 100 base pairs, still more preferably 50 base pairs from adifferentially methylated nucleotide.

As used herein, the term “maternal allele frequency” refers to theratio, represented as a percent, of a maternal allele to the totalamount of alleles present (both paternal and maternal). The term“paternal allele frequency” refers to the ratio, represented as apercent, of a paternal allele to the total amount of alleles present(both paternal and maternal).

As used herein, the term “control DNA sample” or “standard DNA sample”refers to genomic DNA obtained from a healthy individual who does nothave a chromosomal abnormality. Preferably, a control DNA sample isobtained from plasma of a female carrying a healthy fetus who does nothave a chromosomal abnormality. Preferably, one uses a panel of controlsamples. Where certain chromosome anomalies are known one can also havestandards that are indicative of a specific disease or condition. Thus,for example, to screen for three different chromosomal aneuploidies in amaternal plasma of a pregnant female, one preferably uses a panel ofcontrol DNAs that have been isolated from plasma of mothers who areknown to carry a fetus with, for example, chromosome 13, 18, or 21trisomy, and a mother who is pregnant with a fetus who does not have achromosomal abnormality.

The present invention describes a non-invasive approach for diagnosingchromosomal abnormalities that uses fetal DNA obtained from maternalplasma. Fetal DNA comprises approximately 2-6% of the total DNA inmaternal plasma in early and late pregnancy. Theoretically, in a normalfetus, half of the fetal DNA is contributed by the paternally-inheritedfraction.

The present method can be used at any time once pregnancy occurs.Preferably, samples are obtained six weeks or more after conception.Preferably between 6 and 12 weeks after conception.

The technical challenge posed by analysis of fetal DNA in maternalplasma lies in the need to be able to discriminate the fetal DNA fromthe co-existing background maternal DNA. The methods of the presentinvention exploit such differences, for example, the differentialmethylation that is observed between fetal and maternal DNA, as a meansto enrich for the relatively small percentage of fetal DNA present in aplasma DNA sample from the mother. The non-invasive nature of theapproach provides a major advantage over conventional methods ofprenatal diagnosis such as, amniocentesis, chronic vullus sampling andcordocentesis, which are associated with a small but finite risk offetal loss. Also, because the method is not dependent on fetal cellsbeing in any particular cell phase, the method provides a rapiddetection means to determine the presence and also the nature of thechromosomal abnormality.

DNA isolation from blood, plasma, or serum of the pregnant mother can beperformed using any method known to one skilled in the art. Standardmethods of DNA isolation are described, for example, in (Sambrook etal., Molecular Biology: A laboratory Approach, Cold Spring Harbor, N.Y.1989; Ausubel, et al., Current protocols in Molecular Biology, GreenePublishing, Y, 1995). A preferred method for isolation of plasma DNA isdescribed in Chiu et al., 2001, Clin. Chem. 47:1607-1613, which isherein incorporated by reference in its entirety. Other suitable methodsinclude, for example TRI REAGENT® BD (Molecular Research Center, Inc.,Cincinnati, Ohio), which is a reagent for isolation of DNA from, forexample, plasma. TRI REAGENT BD and the single-step method aredescribed, for example, in the U.S. Pat. Nos. 4,843,155 and 5,346,994.

According to the methods of the present invention, fetal DNA can beenriched in a plasma DNA sample that is obtained from an expectingmother by digesting the plasma DNA with one or more enzymes thatselectively cleave part of the maternal DNA. For example, digestingplasma DNA with an enzyme that cleaves only at a DNA recognition sitethat is methylated or by digesting with an enzyme that cleaves only at aDNA recognition site that is unmethylated. Digesting with an enzyme thatcleaves only an unmethylated DNA recognition site will enrich for DNAsequences that are methylated in fetal DNA but are not methylated inmaternal DNA. Alternatively, digesting with an enzyme that cleaves onlya methylated DNA recognition site will enrich for DNA sequences that areunmethylated in fetal DNA but are methylated in maternal DNA. Any enzymethat is capable of selectively cleaving maternal DNA regions and not thecorresponding fetal DNA regions is useful in the present invention.

For example, a CG (or CpG) island is a short stretch if DNA in which thefrequency of the CG sequence is higher than other regions. CpG islandsare frequently found in the promoter regions of genes. Most CpG islandsare more methylated when the gene is inactive and become less methylatedor unmethylated, when the gene is active, i.e. translated. Thus, themethylation pattern is different in different cell types and variesduring development. Since fetal DNA and maternal DNA are likely fromdifferent cell types and from different developmental stage, the regionsof differential methylation can be easily identified and used to enrichthe relative amount of fetal DNA in the maternal plasma sample.

As used herein, “methyl-sensitive” enzymes are DNA restrictionendonucleases that are dependent on the methylation state of their DNArecognition site for activity. For example, there are methyl-sensitiveenzymes that cleave at their DNA recognition sequence only if it is notmethylated. Thus, an unmethylated DNA sample will be cut into smallerfragments than a methylated DNA sample. Similarly, a hypermethylated DNAsample will not be cleaved. In contrast, there are methyl-sensitiveenzymes that cleave at their DNA recognition sequence only if it ismethylated. As used herein, the terms “cleave”, “cut” and “digest” areused interchangeably.

Methyl-sensitive enzymes that digest unmethylated DNA suitable for usein methods of the invention include, but are not limited to, HpaII,HhaI, MaeII, BstUI and AciI. A preferred enzyme of use is HpaII thatcuts only the unmethylated sequence CCGG. Combinations of two or moremethyl-sensitive enzymes that digest only unmethylated DNA can also beused. Suitable enzymes that digest only methylated DNA include, but arenot limited to, DpnI, which cuts at a recognition sequence GATC, andMcrBC, which belongs to the family of AAA⁺ proteins and cuts DNAcontaining modified cytosines and cuts at recognition site 5′ . . .Pu^(m)C(N₄₀₋₃₀₀₀) Pu^(m)C . . . 3′ (New England BioLabs, Inc., Beverly,Mass.).

Cleavage methods and procedures for selected restriction enzymes forcutting DNA at specific sites are well known to the skilled artisan. Forexample, many suppliers of restriction enzymes provide information onconditions and types of DNA sequences cut by specific restrictionenzymes, including New England BioLabs, Pro-Mega Biochems,Boehringer-Mannheim, and the like. Sambrook et al. (See Sambrook et al.,Molecular Biology: A laboratory Approach, Cold Spring Harbor, N.Y. 1989)provide a general description of methods for using restriction enzymesand other enzymes. In the methods of the present invention it ispreferred that the enzymes are used under conditions that will enablecleavage of the maternal DNA with about 95%-100% efficiency, preferablywith about 98%-100% efficiency.

Identification of Methyl-Polymorphic Probes or Markers that DetectDifferent Alleles in Differentially Methylated DNA Regions

The present invention exploits differences in fetal and maternal DNA asa means to enrich for fetal DNA present in a maternal plasma sample.

In one embodiment, the invention exploits differential methylation. Inmammalian cells, methylation plays an important role in gene expression.For example, genes (usually, promoter and first exon regions) arefrequently not methylated in cells where the genes are expressed, andare methylated in cells where the genes are not expressed. Since fetalDNA and maternal DNA in maternal plasma samples are often from differentcell types, and/or of different developmental stages, regions ofdifferential methylation can be identified. DNA fragments whichrepresent regions of differential methylation are then sequenced andscreened for the presence of polymorphic markers, which can be used as“labels” for maternal or paternal allelic DNA. Polymorphic markerslocated in specific genomic regions can be found in public databases,such as NCBI, or discovered by sequencing the differentially methylatedgenomic regions. The identified methyl-polymorphic markers can then beused as a diagnostic marker of chromosomal abnormalities by assessingthe maternal or paternal allele frequency in the maternal plasma sample,wherein the fetal DNA has been enriched according to the methods of thepresent invention. The presence of a ratio other than about ½ of eithermaternal or paternal allele is indicative of either a duplication or adeletion of the particular chromosomal region wherein the polymorphicmarker is located.

Regions of differential methylation can be identified by any means knownin the art and probes and/or primers corresponding to those regions canbe accordingly prepared. Various methods for identifying regions ofdifferential methylation are described in, for example, U.S. Pat. Nos.5,871,917; 5,436,142; and U.S. Patent Application Nos. US20020155451A1,US20030022215A1, and US20030099997, the contents of which are hereinincorporated by reference in their entirety.

Isolation of fetal nucleic acids for the initial purpose of identifyingdifferentially methylated regions in different fetal cells and indifferent fetal developmental stages can be performed from samplesobtained from chorionic villus samples, amniotic fluid samples, oraborted fetuses using methods of nucleic acid isolation well known toone skilled in the art.

Examples of how to identify regions of that are differentiallymethylated in fetal DNA as compared to maternal DNA follow.

One exemplary method is described in U.S. Pat. No. 5,871,917. The methoddetects differential methylation at CpNpG sequences by cutting a testDNA (e.g., fetal DNA) and a control DNA (e.g., maternal DNA) with a CNGspecific restriction enzyme that does not cut methylated DNA. The methoduses one or more rounds of DNA amplification coupled with subtractivehybridization to identify differentially methylated or mutated segmentsof DNA. Thus, the method can selectively identify regions of the fetalgenome that are hypo- or hypermethylated. It is in those regions, onecan then easily identify any polymorphisms, such as SNPs, STRs, orRFLPs, which can be used to detect the allele frequency of the maternaland paternal alleles in the maternal plasma sample wherein fetal DNA isenriched.

In particular, maternal DNA is isolated and compared to DNA isolatedfrom a fetus. The maternal and fetal DNA samples are separately cleavedby a methyl-sensitive enzyme that cleaves only at CNG sites that areunmethylated. The samples are further cleaved with a second enzyme thatcleaves the DNA into a size and complexity appropriate for DNAamplification and subtractive hybridization. Preferably, the secondenzyme cleaves DNA to produce ends that are neither homologous norcomplimentary to a sticky-end produced by the methyl-sensitive enzyme.After cleavage, a set of adaptors is ligated onto the sticky-endsproduced by the CNG specific restriction enzyme that does not cutmethylated DNA. The adaptors are selected so that they will ligate tothe CG-rich-ends of the DNA cut by the methyl-sensitive enzyme but notto the ends of the fragments that were cut with the second enzyme. Theadaptors are chosen not only to ligate to DNA-ends cut by themethyl-sensitive enzyme, but also to be a good size and DNA sequence toact as a recognition site for primers to be used in DNA amplification.Only those fragments that have the adaptor and thus were cut with themethyl-sensitive enzyme will be amplified in a PCR reaction usingadaptor sequence primers.

The two samples are separately amplified. After amplification, the firstset of adaptors are removed from the ends of the amplified fragments bycleavage with the methyl-sensitive enzyme. This preserves the originalends of the fragments.

A second set of adaptors are ligated to the amplified maternal DNA, butnot the amplified fetal DNA. The second set of adaptors is selected sothat they do not have the same sequence as the first set of adaptors andso that they ligate only to DNA-ends cut by the methyl-sensitive enzyme.The second set of adaptors also provides a good recognition site forprimers which are used for amplification.

At least one round of subtraction/hybridization followed DNAamplification is performed by standard methods. The result is aselection of DNA fragments that are uniquely unmethylated in maternalDNA, which can be used as probes to detect identified sites ofmethylation in the fetal genome.

In particular, the maternal DNA is mixed with a large excess of fetalDNA as described in U.S. Pat. No. 5,871,917. The subtractionhybridization mixture is then amplified by in vitro DNA amplificationprocedures using primers that hybridize to the second adaptor-ends.Thus, only maternal DNA fragments with second adaptor ends areamplified. Any maternal DNA that is hybridized to fetal DNA will not beamplified. A large excess of maternal DNA is used to promote formationof hybrids that are commonly found in both the fetal and maternalsamples. The result is isolation of umethylated maternal DNA fragmentsthat are uniquely methylated in fetal DNA. Fragments are isolated bystandard methods known in the art.

A Southern Blot Hybridization can be performed to confirm that theisolated fragments detect regions of differential methylation. Maternaland fetal genomic DNA can be cut with a methyl-sensitive enzyme andhypometylation or hypermethylation at a specific site can be detected byobserving whether the size or intensity of a DNA fragment cut with therestriction enzymes is the same between samples. This can be done byelectrophoresis analysis and hybridizing the probe to the maternal andfetal DNA samples and observing whether the two hybridization complexesare the same or different sizes and/or intensities. Detailed methodologyfor gel electrophoretic and nucleic acid hybridization techniques arewell known to one skilled in the art and protocols can be found, forexample, in Sambrook et al., Molecular Biology: A laboratory Approach,Cold Spring Harbor, N.Y. 1989.

The fragment sequences can then be screened for polymorphic markers thatcan be used to differentiate between paternal or maternal alleles, whichcan be used as methyl-polymorphic probes as described herein. Probesisolated by the technique described above have at least 14 nucleotidesto about 200 nucleotides.

Examples of suitable restriction enzymes for use in the above methodinclude, but are not limited to BsiSI, Hin2I, MseI, Sau3A, RsaI, TspEI,MaeI, NiaIII, DpnI and the like. A preferred methyl-sensitive enzyme isHpa II that recognizes and cleaves at nonmethylated CCGG sequences butnot at CCGG sequences where the outer cytosine is methylated.

Differential methylation can also be assessed by the methods describedin U.S. Patent Application No. 2003009997, which discloses a method fordetecting the presence of differential methylation between two sourcesof DNA using enzymes that degrade either unmethylated or methylated DNA.For example, genomic maternal DNA can be treated with a mixture ofmethyl-sensitive enzymes that cleave only unmethylated DNA, such asHpaII, HhaI, MaeI, BstUI, and AciI so as to degrade unmethylated DNA.Genomic fetal DNA can then be treated with an enzyme that degradesmethylated DNA, such as McrBC (New England Biolabs, Inc.). Subtractivehybridization then permits selective extraction of sequences that aredifferentially methylated between fetal and maternal DNA.

Alternatively, differential methylation between maternal and fetal DNAcan be assessed by bisulfide treatment followed by either 1) sequencing,or 2) base-specific cleavage followed by mass spectrometric analysis asdescribed in von Wintzingerode et al., 2002, PNAS, 99:7039-44, hereinincorporated by reference in its entirety.

To serve as a probe, the identified methyl-polymorphic markers can belabeled by any procedure known in the art, for example by incorporationof nucleotides linked to a “reporter molecule”.

A “reporter molecule”, as used herein, is a molecule which provides ananalytically identifiable signal allowing detection of a hybridizedprobe. Detection may be either qualitative or quantitative. Commonlyused reporter molecules include fluorophores, enzymes, biotin,chemiluminescent molecules, bioluminescent molecules, digoxigenin,avidin, streptavidin, or radioisotopes. Commonly used enzymes includehorseradish peroxidase, alkaline phosphatase, glucose oxidase andbeta-galactosidase, among others. Enzymes can be conjugated to avidin orstreptavidin for use with a biotinylated probe. Similarly, probes can beconjugated to avidin or streptavidin for use with a biotinylated enzyme.The substrates to be used with these enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable color change. For example, p-nitrophenyl phosphate issuitable for use with alkaline phosphatase reporter molecules; forhorseradish peroxidase, 1,2-phenylenediamine, 5-aminosalicylic acid ortolidine are commonly used. Incorporation of a reporter molecule into aDNA probe can be by any method known to the skilled artisan, for exampleby nick translation, primer extension, random oligo priming, by 3′ or 5′end labeling or by other means (see, for example, Sambrook et al.Molecular Biology: A laboratory Approach, Cold Spring Harbor, N.Y.1989).

Alternatively, the identified methyl-polymorphic markers need not belabeled and can be used to quantitate allelic frequency using a massspectrometry technique described in Ding C. and Cantor C. R., 2003,Proc. Natl. Acad. Sci. U.S.A. 100, 3059-64, which is herein incorporatedby reference in its entirety.

Comparing Maternal and Paternal Allele Frequency

To diagnose the presence of a chromosomal abnormality using maternalplasma DNA according to the methods of the present invention, the plasmaDNA can be first enriched for fetal DNA by digestion of plasma DNA withenzymes that selectively cleave maternal DNA, for example by usingenzymes sensitive to the methylation state of the DNA. Polymorphicmarkers, such as the methyl-polymorphic markers described herein whichare adjacent to or within differentially methylated fetal DNA regions,can be used to determine the allele frequency of either a paternal or amaternal allele. The allele frequency is compared to the allelefrequency present in a control DNA sample (e.g. a genomic DNA obtainedfrom an individual that does not have a chromosomal abnormality).Preferably, the control DNA is isolated from the plasma of a femalepregnant with a healthy fetus.

A difference in allele frequency is indicative that a chromosomalabnormality is present in fetal DNA. Thus, in a normal sample, whereinsubstantially all of the maternal DNA has been digested, a ratio ofmaternal and paternal allele in any given locus is about 12 or 50% ofthe alleles present are of maternal and 50% of paternal origin. If anylocus is either duplicated or deleted because of partial or completechromosome duplication or deletion of a region wherein the particularallele is present, the ratio will differ from the 50%:50% ratio. Thechromosomal abnormality can be a DNA deletion or duplication thatincludes the DNA sequence detected by the polymorphic probe. Thedeletion or duplication can be the result of chromosome aneuploidy (thepresence or absence of an entire chromosome) or it can be the result ofdeletion or duplication within a chromosome. Chromosomal aneuploidy canbe confirmed by any method known to those skilled in the art. Apreferred method for conformation of chromosomal aneuploidy isamniocentesis followed by fluorescence in situ hybridization (FISH), bytraditional karyotyping with Geisma-staining or by spectral karyotyping(SKY), which are all methods well known to one skilled in the art.

Any polymorphic marker located in the region with, for example thedifferential methylation status between maternal and fetal DNA, or anyother, preferably epigenetic information difference, between thematernal and fetal DNA, can be used to detect the frequency of thematernal and paternal allele in the fetal DNA present in the maternalplasma. Thus, once the differentially methylated regions have beendetermined, a skilled artisan can easily turn to databases, wherein one,and preferably more than one, SNPs or other polymorphic markers can bepicked that are located within the differentially methylated DNAregions. Alternatively, sequencing the region from several individualscan reveal new useful nucleic acid polymorphisms.

Methods for determining allele frequency are well known to those skilledin the art. The allelic frequency using the polymorphic probes thatdetect differential DNA regions, can be determined by any such method.For example, a quantifiable label can be incorporated intomethyl-polymorphic probes that specifically detect either maternal ofpaternal DNA. The probes are then hybridized to the DNA sample, e.g., bySouthern Blot, and quantitated. Preferred labels for such a method areradioisotopes and fluorescent markers that can be quantitated bydensitometry.

After digestion of the maternal nucleic acids in the plasma sample, thematernal and paternal alleles present in the enriched fetal nucleic acidsample are preferably amplified using PCR. The allele ratio is thenmeasured using various differential amplification methods describedbelow, including different primer extension methods. Preferably, theanalysis is performed using a primer-extension reaction after apolymerase chain reaction (PCR) and detecting the primer extensionproducts using mass spectrometry. One preferred method of the presentinvention for determination of allelic frequency using mass spectrometrytechnique is described in Ding C. and Cantor C. R., 2003, Proc. Natl.Acad. Sci. U.S.A. 100, 3059-64. The MassARRAY system is based onmatrix-assisted laser desorption ionization/time-of-flight (MALDI-TOF)mass spectrometric (MS) analysis of primer-extension products (Tang, K.et al. Proc Natl Acad Sci USA 96, 10016-10020 (1999)).

Alternatively, the detection can be performed using, for example,electrophoretic methods including capillary electrophoresis, usingdenaturing high performance liquid chromatography (D-HPLC), using anInvader® Assay (Third Wave Technologies, Inc., Madison, Wis.),pyrosequencing techniques (Pyrosequencing, Inc., Westborough, Mass.) orsolid-phase minisequencing (U.S. Pat. No. 6,013,431, Suomalainen et al.Mol. Biotechnol. June;15(2):123-31, 2000).

The allele frequency is presented as the ratio of either a maternalallele and a paternal allele in the total amount of alleles present(both paternal and maternal). Since the allelic frequency is a ratio,the maternal or paternal allele frequency of the control DNA sample canbe determined using either different or the same probes used to detectthe allele frequency in fetal DNA.

Preferably, two, three, four, 5-10 or even more than 10 polymorphic locican be analyzed in the same reaction. Using a pool of severalpolymorphic markers allows one to perform the analysis even if theparental alleles are not known and still allow identification of atleast one informative marker, i.e., loci wherein the two alleles in thefetal sample are different, i.e. the allele inherited from the father isdifferent than the allele inherited from the mother. Preferably, themarkers are selected in different locations along the desiredchromosomes, such as chromosomes 21, 13, and 18.

Alternatively, one can first determine the informative loci bygenotyping the maternal and paternal loci using a selected polymorphicmarkers in the chromosomal regions that are differentially methylated inthe maternal and fetal DNA, and use only a selection of those markers,wherein the alleles differ, in determining the allele frequency in thefetal DNA sample.

Herein, a difference in allelic frequency of either maternal or paternalalleles refers to a difference that is at least 3%, preferably at least10%, and more preferably at least 15%. Preferably, the normal alleleratio of maternal and paternal alleles in the plasma DNA sample, whereinthe maternal DNA has been substantially completely digested is 50% ofmaternal allele and 50% of paternal allele. If this allelic ratiochanges for any give locus, the fetus is likely to carry a duplicationor deletion of the chromosomal region, wherein the allele is located.

In one embodiment of the invention, an amplification step is performedto further enrich for fetal DNA in a sample of maternal plasma.Amplification is performed after the enrichment of fetal DNA in plasmaDNA by enzymatic digestion and prior to the detection of allelicfrequency/ratio. Amplification can be performed by any method known inthe art (such as, Polymerase Chain Reaction (PCR) or rolling circleamplification) using primers that anneal to the selected fetal DNAregions. Oligonucleotide primers are selected such that they anneal tothe sequence to be amplified. Preferably, the amplification is performedusing the rolling circle method which allows combining the amplificationreaction with an enzymatic methylation step, wherein the methylationstatus of the fetal and/or the remaining maternal DNA is preservedthrough the amplification. Preferably, the amplification step isfollowed by another enzymatic digestion step to further remove anyremaining maternal DNA from the sample.

Oligonucleotide primers for PCT, rolling circle amplification and primerextension reactions described herein, may be synthesized using methodswell known in the art, including, for example, the phosphotriester (seeNarang, S. A., et al., 1979, Meth. Enzymol., 68:90; and U.S. Pat. No.4,356,270), phosphodiester (Brown, et al., 1979, Meth. Enzymol.,68:109), and phosphoramidite (Beaucage, 1993, Meth. Mol. Biol., 20:33)approaches. Each of these references is incorporated herein in itsentirety by reference.

Alternatively, one may mask the maternal DNA and/or selectively amplifythe fetal DNA to accentuate the amount of fetal DNA in the sample andallow detection of allele ratio in the fetal DNA.

In one aspect, the invention provides a method for prenatal diagnosis ofchromosomal abnormality in a fetus. The method comprises the steps of a)obtaining a plasma/blood/serum sample from a pregnant female andisolating DNA from said sample, b) digesting isolated DNA with amethyl-sensitive enzyme that digests only unmethylated DNA, c) isolatingundigested DNA from step b), d) amplifying the undigested DNA from stepc) while simultaneously using a DNA methylase to methylate nascenthemi-methylated DNA, e) digesting amplified DNA of step d) with amethyl-sensitive enzyme that digests only unmethylated DNA, f)determining the paternal or maternal allele frequency using polymorphicmarkers adjacent to unmethylated fetal DNA regions; and, g) comparingthe paternal or maternal allele frequency or ratio of step f) to acontrol DNA sample, wherein a difference in allele frequency isindicative of a chromosomal abnormality in the fetus.

The first digestion of the maternal DNA sample enriches for fetal DNAthat is methylated. The amplification step provides for additionalenrichment of fetal DNA by amplifying and further maintaining themethylation status of fetal DNA.

The amplification step is combined with the use of a DNA methylase thatis specific for hemi-methylated DNA (such as, Dnmt1) in order tomethylate nascent hemi-methylated DNA. Since fetal DNA that ismethylated is enriched in the first digestion, the methylase will onlymethylate fetal DNA and not maternal DNA in the amplification process.During amplification, methylated fetal DNA and any backgroundunmethylated maternal DNA are produced. Therefore, the amplified DNAsample is again digested with a methyl-sensitive enzyme that digestsonly unmethylated DNA. Such an amplification procedure provides a secondstage of fetal DNA enrichment.

In a preferred embodiment, rolling circle amplification (RCA) is used.Rolling circle amplification is an isothermal process for generatingmultiple copies of a sequence. In rolling circle DNA replication invivo, a DNA polymerase extends a primer on a circular template (Komberg,A. and Baker, T. A. DNA Replication, W.H. Freeman, New York, 1991). Theproduct consists of tandemly linked copies of the complementary sequenceof the template. RCA is a method that has been adapted for use in vitrofor DNA amplification (Fire, A. and Si-Qun Xu, Proc. Natl. Acad. Sci.USA, 1995, 92:4641-4645; Lui, D., et al., J. Am. Chem. Soc., 1996,118:1587-1594; Lizardi, P. M., et al., Nature Genetics, 1998,19:225-232; U.S. Pat. No. 5,714,320 to Kool). RCA techniques are wellknown in the art, including linear RCA (LRCA). Any such RCA techniquecan be used in the present invention.

The methods of the present invention are suitable for diagnosing achromosomal abnormality in a fetus, e.g., detecting chromosomaldeletions, duplications and/or aneuploidy.

The advantage of the methods described herein, is that chromosomalabnormalities can be detected using plasma/blood/serum DNA from themother, which contains only a small percent of fetal cells and hence, asmall percentage of fetal DNA. The present invention provides methodsfor the enrichment of fetal DNA through the specific digestion ofmaternal DNA and provides an easy non-invasive approach to obtainingfetal DNA samples that can be used to screen for chromosomalabnormalities in fetuses carried by pregnant females.

Moreover, because the method does not rely on visual inspection ofchromosomes, the need to grow fetal cells and/or synchronize the cellcycle of the fetal cells is not needed thus allowing rapid screening inthe time sensitive prenatal diagnosis.

The methods are particularly useful for, but not limited to, diagnosingchromosomal aneuploidies such as Down's syndrome, Turner's syndrome,trisomy 13, trisomy 18, and Klinefelter syndrome.

Down's syndrome is characterized by the presence of 3 copies chromosome21 instead of one, and is often referred to as trisomy 21. Three to fourpercent of all cases of trisomy 21 are due to RobertsonianTranslocation. In this case, two breaks occur in separate chromosomes,usually the 14th and 21 st chromosomes. There is rearrangement of thegenetic material so that some of the 14th chromosome is replaced byextra 21st chromosome. So while the number of chromosomes remain normal,there is a triplication of the 21st chromosome material. Some of thesechildren may only have triplication of part of the 21 st chromosomeinstead of the whole chromosome, which is called a partial trisomy 21.The extra DNA produces the physical and mental characteristics of Downsyndrome, which include a small head that is flattened in the back;slanted eyes; extra skin folds at the corners of the eyes; small ears,nose and mouth; short stature; small hands and feet; and some degree ofmental disability.

Trisomy 13 and 18 refer to an extra chromosome 13 or 18, respectively.Trisomy 13, also known as Patua's syndrome, is characterized by a smallat birth weight. Spells of interrupted breathing (apnea) in earlyinfancy are frequent, and mental retardation is usually severe. Manyaffected children appear to be deaf. A moderately small head(microcephaly) with sloping forehead, wide joints and openings betweenparietal bones of the head are present. Gross anatomic defects of thebrain, especially failure of the forebrain to divide properly(holoprosencephaly) are common. A hernial protrusion of the cord and itsmeninges through a defect in the vertebral canal (myelomeningocele) isfound in almost 50% of cases.

The entire eye is usually small (microphthalmia), and a defect of theiris tissue (coloboma), and faulty development of the retina (retinaldysplasia) occur frequently. The supraorbital ridges are shallow andpalapebral fissures are usually slanted. Cleft lip, cleft palate, brboth are present in most cases. The ears are abnormally shaped andunusually low-set.

Trisomy 18, or Edwards syndrome, results in babies that appear thin andfrail. They fail to thrive and have problems feeding. Trisomy 18 causesa small head size, with the back of the head (occiput) prominent. Earsare usually low set on the head. The mouth and jaw are unusually small,and there is a shortened sternum (breastbone). At birth, these babiesare small for their age, even when delivered full-term, and have a weakcry. Their response to sound is decreased and there is often a historyof infrequent fetal activity during the pregnancy. About 90 percent ofbabies with trisomy 18 have heart defects. They clench their fists in acharacteristic manner and extending the fingers fully is difficult.Joint contractures, where the arms and legs are in a bent positionrather than relaxed, are usually present. The feet may be referred to as“rocker bottom” due to their shape. Babies with trisomy 18 may also havespinal bifida (in 6 percent of cases), eye problems (in 10 percent ofcases), cleft lip and palate (in most cases), and hearing loss (in mostcases). It is also common to see feeding problems, slow growth, seizures(about 30 percent of cases in the first year), high blood pressure,kidney problems and scoliosis (curvature of the spine). In males, thetestes fail to descend into the scrotum.

Turner syndrome, or monosomy X, is usually caused by a missing Xchromosome. It affects 1 out of 3,000 live births. The main features ofthe syndrome are short stature, webbing of the skin of the neck, absentor retarded development of secondary sexual characteristics, absence ofmenstruation, coarctation (narrowing) of the aorta, and abnormalities ofthe eyes and bones. The condition is usually either diagnosed at birthbecause of the associated anomalies, or at puberty when there is absentor delayed menses and delayed development of normal secondary sexualcharacteristics. The methods described herein enable pre-birthdiagnosis.

Klinefelter syndrome refers to males that have an extra sex chromosome,XXY instead of the usual male arrangement, XY. The syndrome ischaracterized by men who had enlarged breasts, sparse facial and bodyhair, small testes, and an inability to produce sperm. Although they arenot mentally retarded, most XXY males also have some degree of languageimpairment.

The methods of the present invention, provide for a non-invasiveapproach for diagnosis of chromosomal abnormalities and relatedsyndromes.

The invention will now be further illustrated with reference to thefollowing examples. It will be appreciated that what follows is by wayof example only and that modifications to detail may be made while stillfalling within the scope of the invention.

EXAMPLES

The following is an example that illustrates the steps for diagnosis ofDown's syndrome using maternal plasma DNA. The approach is applicablefor any chromosomal aneuploidy or chromosomal DNA duplication.

In Down's syndrome, the fetus has three chromosomes 21. In 90% of thecases of trisomy 21, the fetus obtained two chromosomes 21 from themother and one chromosome 21 from the father. The detection of extrachromosome 21 DNA is performed as follows:

DNA regions in chromosome 21 are screened for differentialmethylation—methylated in fetal DNA and not methylated in maternal DNA(mostly peripheral blood cells).

Polymorphic markers that are close to the differentially methylated DNAregions that can be used as labels for maternal and paternal DNA areidentified.

A plasma sample is obtained from a pregnant female and DNA is isolatedfrom the sample.

The isolated plasma DNA is treated with a methylation sensitive enzyme(e.g. Hpa II) that cuts only the unmethylated DNA sequence CCGG. TheEnzyme is used to digest unmethylated maternal DNA, leaving onlymethylated fetal DNA fragments. Alternatively, an enzyme that cuts onlymethylated DNA (such as Dpn I, which recognizes the sequence GATC) canalso be used, the following steps are adjusted accordingly.

A substantial difference between the paternal allele frequency intrisomy 21 and the paternal allele frequency for a normal individual(table below) will be observed. In the table, allele A is maternalspecific and allele B is paternal specific.

In the table below, the paternal allele frequency less than the controlis indicative of Down's syndrome. Maternal Paternal Allele DNA Fetal DNAfrequency Trisomy 21 AA AAB ⅓ or 33.3% Control AA AB ½ or 50% sample

Confirmation that the chromosomal abnormality is due to chromosomalaneuploidy represented by an extra chromosome 21 can be confirmed bymeans known in the art, such as amniocentesis.

If the enzymatic digestion is less than 100% efficient, a difference inallele frequency will still be observed but instead of a 16.7%difference (as illustrated in table), the difference observed may be inthe range of 5-10%, and it is thus preferable, that a control locus (C/Dalleles in the normal, or non-aneuploidy locus in the table below) isused to provide a control. This is illustrated in the following table:Maternal Fetal B (or D) % B (or D) % DNA DNA 100 digestion 98% digestionTrisomy 21 AA AAB 33.3% 20% Non-aneuploidy CC CD   50% 25%

In the case that there is not 100% digestion, an additionalamplification scheme can also be incorporated in to the method describedabove in order to further enrich for fetal DNA. Assuming a DNA regionthat is methylated in fetal DNA and not methylated in maternal DNA isused.

The majority of maternal DNA will be digested using a methylationsensitive enzyme as described above. This is the first step of fetal DNAenrichment.

Both maternal and fetal DNA will then be amplified by an isothermalmechanism (such as rolling circle amplification). Simultaneously, a DNAmethylase (such as Dnmt1) specific for hemi-methylated DNA is used tomethylate nascent hemi-methylated DNA. Since only the fetal DNA ismethylated at the beginning, the methylase will only methylate fetal DNAin the amplification process. As a result, methylated fetal DNA andunmethylated DNA are produced.

The amplified sample will then be digested again using a methylationsensitive enzyme that will digest the unmethylated maternal DNA (e.g.HpaII). This is the second step of fetal DNA enrichment. After thisstep, vast majority of DNA left is fetal DNA. For future DNAquantifications, this is equivalent to 100% Hpa II digestion.

All references described herein are incorporated herein by reference.

1. A method for prenatal diagnosis of chromosomal abnormality in apredetermined DNA region comprising the steps of: a) obtaining a plasmasample from a pregnant female; b) digesting DNA from said plasma samplewith an enzyme that selectively and substantially completely digests thematernal DNA to obtain a DNA sample enriched for fetal DNA regions; andc) determining the paternal or maternal allele frequency usingpolymorphic markers adjacent to or within the fetal DNA regions in thesample of step (b), wherein a difference in allele frequency from otherthan 50% of paternal and 50% of maternal allele as compared to a normalcontrol, which does not comprise a chromosomal abnormality is indicativeof a chromosomal abnormality.
 2. The method of claim 1, wherein the DNAis isolated from the plasma sample before it is digested.
 3. The methodof claim 1, wherein comparing the paternal or maternal allele frequencyof step (c) is performed against at least one internal control locatedin a chromosome, duplication or deletion of which is not a target ofdiagnosis, and wherein both maternal and paternal alleles are present inequal amount, wherein deviation of the ratio from the internal controlindicates presence of chromosomal abnormality.
 4. The method of claim 1further comprising a DNA amplification step performed after step (a) andbefore step (c).
 5. The method of claim 1, wherein said enzyme of step(b) is a methyl-sensitive enzyme.
 6. The method of claim 5, wherein saidmethyl-sensitive enzyme digests only at DNA recognition sites that areumethylated and wherein the maternal or paternal allele frequency isdetermined using polymorphic markers adjacent to or within methylatedfetal DNA regions.
 7. The method of claim 3, wherein saidmethyl-sensitive enzyme digests only at DNA recognition sites that aremethylated, and wherein the maternal or paternal allele frequency isdetermined using polymorphic markers adjacent to or within unmethylatedfetal DNA regions.
 8. A method for prenatal diagnosis of chromosomalabnormality comprising the steps of: a) obtaining a plasma sample from apregnant female; b) digesting nucleic acids present in said plasmasample with a methyl-sensitive enzyme that digests only unmethylatedDNA; c) optionally isolating undigested nucleic acid from step (b); d)amplifying the undigested nucleic acid from step (b) or (c) while usinga nucleic acid methylase to methylate nascent hemi-methylated nucleicacid; e) digesting amplified nucleic acid of step (d) with amethyl-sensitive enzyme that digests only unmethylated nucleic acid; andf) determining the paternal or maternal allele frequency usingpolymorphic markers adjacent to or within unmethylated fetal nucleicacid regions, wherein a difference in allele frequency other than 50% ofmaternal and 50% of paternal is indicative of a chromosomal abnormality.9. The method of claim 8, wherein the comparing of the paternal ormaternal allele frequency of step (f) is performed against to a controlnucleic acid sample, wherein a difference of other than the ratio in thecontrol sample is indicative of a chromosomal abnormality.
 10. Themethod of claim 8, wherein the nucleic acid is DNA.
 11. The method ofclaim 8, wherein the nucleic acid is isolated from the plasma samplebefore it is digested.
 12. The method of claim 1 or 8, wherein thechromosomal abnormality is DNA duplication.
 13. The method of claim 1 or8, wherein the chromosomal abnormality is a DNA deletion.
 14. The methodof claim 1 or 8, wherein the chromosomal abnormality is aneuploidy. 15.The method of claim 14, wherein said aneuploidy is selected from thegroup consisting of trisomy 21, trisomy 18, and trisomy
 13. 16. A methodof diagnosing fetal chromosomal abnormality comprising the steps of: a)obtaining a plasma sample from a pregnant female; b) selectivelytreating said plasma sample to enrich the sample for at least one fetalnucleic acid region; c) determining the paternal or maternal allelefrequency using at least one polymorphic marker adjacent to or withinthe at least one fetal nucleic acid region in the sample of step (b);and d) comparing the paternal or maternal allele frequency of step (c)to a control DNA sample, wherein a difference in allele frequency fromother than 50% of paternal and 50% of maternal allele is indicative of achromosomal abnormality.
 17. A method of diagnosing fetal chromosomalabnormality comprising the steps of: a) obtaining a plasma sample from apregnant female; b) selectively treating said plasma sample to enrichthe sample for at least one fetal nucleic acid region; c) determiningthe paternal or maternal allele frequency using at least one polymorphicmarker adjacent to or within the at least one fetal nucleic acid regionin the sample of step (b); and d) comparing the paternal or maternalallele frequency of step (c) to a control DNA sample wherein thematernal and paternal alleles are present in predetermined amounts,wherein a difference in allele frequency from other than 50% of paternaland 50% of maternal allele as compared to the control is indicative of achromosomal abnormality.
 18. A kit for prenatal diagnosis of chromosomalabnormalities comprising a methylation-sensitive enzyme, at least onepair of nucleic acid amplification primers capable of annealing and thusamplifying regions flanking sites that contain at least one polymorphiclocus within differentially methylated regions in fetal and maternal DNApresent in maternal plasma, at least one primer or probe to allowdetection of alleles in the at least one polymorphic locus, and aninstruction manual instructing the user to perform the steps of taking aplasma sample from a pregnant female, selectively digesting the nucleicacids present in said plasma sample with the methylation-sensitiveenzyme to enrich the fetal nucleic acids in the sample, performingnucleic acid amplification using the amplification primers and detectingthe alleles present in the sample enriched for the fetal nucleic acids,and interpreting the results so that if the ratio of two differentalleles in the locus deviates from a control wherein the alleles arepresent in equal amounts, the fetus is affected with a chromosomalabnormality.
 19. The kit of claim 18, further comprising a controlnucleic acid panel, wherein the controls comprise nucleic acids isolatedfrom females pregnant with fetuses carrying known chromosomalabnormalities and females pregnant with fetuses without chromosomalabnormalities.
 20. The kit of claim 19, further comprising an internalcontrol of at least one pair of amplification primers and a detectionprimer or probe, wherein the primers and/or probe are selected from anucleic acid region that is differentially methylated in fetal andmaternal DNA present in maternal plasma, but that occur in chromosomes,wherein duplication or deletion is rare, so as to provide an internalcontrol.
 21. The kit of claim 19, wherein the prenatal diagnosis is forchromosome 13, 18 or 21 duplications and the internal control is locatedin any other autosome than 13, 18, or 21.